1. Field of the Invention
The present invention generally relates to a method for the large-scale production of human serum butyrylcholinesterase (HuBChE) from Cohn Fraction IV-4 paste.
2. Description of the Related Art
Exposure to organophosphorus compounds (OPs) in the form of nerve agents and pesticides poses an ever increasing threat to military and civilian populations. The serious medical challenges posed by chemical warfare agents to both the military and civilian health care systems became evident in the Iran-Iraq conflict and the 1995 Tokyo subway incident, respectively. The acute toxicity of OPs is usually attributed to their irreversible inhibition of acetylcholinesterase (AChE). The resultant increase in acetylcholine concentration manifests at the cholinergic synapses of both the peripheral and central nervous systems, which precipitates a cholinergic crisis characterized by miosis, increased tracheobronchial and salivary secretions, bronchoconstriction, bradycardia, fasciculations, behavioral incapacitation, muscular weakness, and convulsions, ultimately culminating in death by respiratory failure. Current antidotal regiments for OP poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and post-exposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride. See Gray (1984) Drug Metab. Rev. 15:557-589. Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits or in many cases, permanent brain damage. See Dirnhuber et al. (1979) J. Pharm. Pharmacol. 31:295-299; McLeod (1985) Fundam. Appl. Toxicol. 5:S10-S16; and Dunn & Sidell (1989) J. Am. Med. Assoc. 262:649-652. These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets and prevent the in vivo toxicity of OPs and post exposure incapacitation.
Among the enzymes examined as potent scavengers of highly toxic OP nerve agents, significant advances have been made using ChEs. Exogenous administration of plasma-derived ChEs such as AChE from fetal bovine serum (FBS) and BChE from human and equine serum (Eq), in both rodent and non-human primate models, has been successfully used as a safe and efficacious prophylactic treatment to prevent poisoning by OP compounds. See Doctor et al. (2001) “New approaches to Medical protection against chemical warfare nerve agents” CHEMICAL WARFARE AGENTS: TOXICITY AT LOW LEVELS, NYC, CRC Press, pp. 191-214. In contrast to the currently used multi-drug treatment, ChEs as prophylactic agents have the advantage of being single pretreatment scavengers capable of protecting against multiple LD50's of a wide variety of potent OPs without the requirement of additional post-exposure therapy.
Of the ChEs evaluated so far, HuBChE has several advantages as an exogenously administered prophylactic for human use. See Ashani (2000) Drug Dev. Res. 50:298-308. First, it reacts rapidly with all highly toxic OPs, offering a broad range of protection for nerve agents including, soman, sarin, tabun, and VX. Studies in mice, rats, guinea pigs and rhesus monkeys clearly demonstrated that HuBChE could function as an antidote for all OP nerve agents. See Raveh et al. (1993) Biochem. Pharmacol. 45:2465-2474; Brandeis et al. (1993) Pharmacol. Biochem. Behav. 46:889-896; Allon et al. (1998) Toxicol. Sci. 43:121-128; and Raveh et al. (1997) Toxicol. Pharmacol. 145:43-53. These studies also showed that pretreatment with HuBChE was effective in preventing mortality as well as development of behavioral deficits without the need for additional post-exposure therapy. Second it possesses a very long retention time in human circulation and is readily absorbed from sites of injection. Although the reported values of half-life of exogenously administered HuBChE in humans vary from 3.4 to 11 days, they suggest that the circulatory stability of the enzyme is sufficient for its use as a pretreatment drug. See Ostergaard et al. (1988) Acta Anaesthesiologica Scandinavica 32:266-269. The extended stability of exogenously administered HuBChE was also demonstrated in mice and rats, guinea pigs, and rhesus monkeys. These results suggest that a single injection of HuBChE will provide long-lasting protection if used as a prophylactic treatment. Third, since the enzyme is from a human source, it should not produce any adverse immunological responses upon repeated administration into humans. The lack of reports indicating untoward side-effects in humans following plasma transfusions and i.v. injections of partially purified HuBChE support our contention. See Cascio et al. (1988) Minerva Anestesiol. 54:337-338. Similarly, the exogenous administration of 13-20 mg/kg doses of HuBChE did not seem to affect gross behavior in mice, rats or guinea pigs and no behavior alterations were reported in rhesus monkeys treated with 13-34 mg of HuBChE.
Extrapolation of data obtained from prophylaxis experiments with HuBChE in four species suggests that a dose of 200 mg of HuBChE as a prophylactic treatment can protect humans from exposure of up to 2 LD50 of soman. Smaller doses of 50 mg of enzyme would be sufficient to provide protection against low-level exposure to nerve agents. In addition to its use as a pretreatment for a variety of wartime scenarios, including covert actions, it also has potential use for first responders (civilians) reacting to intentional/accidental nerve gas release or pesticide overexposure. In addition, since HuBChE catalyzes the hydrolysis of cocaine and short-acting muscle relaxants succinylcholine and mivacurium, it could be an effective treatment for cocaine intoxication, as well as succinylcholine- and mivacurium-induced apnea.
The foremost requirement to advance HuBChE as a bioscavenger for human use was to obtain sufficient amounts of purified enzyme for conducting animal and clinical studies. Although a procedure for the purification of HuBChE from human plasma, which contains about 2 mg of enzyme per liter of plasma, was described, this source is not suitable for producing large quantities of HuBChE for clinical and commercial uses.
Thus a need exists for methods for the large-scale production of HuBChE.